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In JoVE (1)
Other Publications (5)
Articles by Adi Salzberg in JoVE
JoVE Neuroscience
Visualization of Proprioceptors in Drosophila Larvae and Pupae
A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.
Other articles by Adi Salzberg on PubMed
Identification of Methylated Sequences in Genomic DNA of Adult Drosophila Melanogaster
The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing.
Additional Sex Combs Affects Antennal Development by Means of Spatially Restricted Repression of Antp and Wg
Additional sex combs (Asx) is thought to function in protein complexes of both the Trithorax and Polycomb groups, but very little is known about its developmental roles. Here, we present a detailed analysis of Asx's role in antennal development. We show that loss of Asx in the antennal disc causes a complex phenotype, which consists of distal antenna-to-leg transformations and outgrowth of ectopic leg-like appendages from the Dpp-expressing domain of the disc. Our analyses suggest that these phenotypes are caused mainly by segment-specific de-repression of Antp and expansion of wg expression. We thus conclude that Asx functions normally to repress Antp and to restrict wg expression in specific regions of the developing disc. We also show that, in the absence of Asx's function, Antp expression does not lead to efficient repression of the antennal-determining gene hth, suggesting that Asx is also required for the repression of hth by Antp.
Chromosomal Binding Sites of the Homeotic Cofactor Homothorax
The Meis family oncoproteins play a crucial role in leukemogenesis and are highly expressed in other types of cancer as well. The transforming potential of Meis proteins depends on their ability to activate gene expression and therefore, revealing the identity of their target genes is very important. The genome of the fruit fly Drosophila melanogaster contains a single Meis gene, homothorax (hth), which plays multiple roles in embryonic and adult development. Mutations in hth affect the development of numerous embryonic and adult tissues, suggesting that Hth regulates the transcription of a large number of genes. However, it is not known how many genes are regulated directly by Hth and what is the nature of these genes. To address this question, we examined the distribution of the in vivo binding sites of Hth on polytene chromosomes. We found that in the salivary glands (SG) of third instar larvae, Hth binds to approximately 150 chromosomal sites in a very reproducible pattern. More than hundred of these sites were mapped cytologically. Interestingly, Hth accumulates at high levels in some of the most prominent hormone-induced chromosomal puffs, pointing to a possible role of Hth in activation of ecdysone-induced targets. Interfering with the normal transcriptional activity of Hth in larval SGs leads to dramatic reduction in cell size and DNA content implicating Hth in the regulation of cell growth and endoreplication in larval SGs.
The Proprioceptive and Contractile Systems in Drosophila Are Both Patterned by the EGR Family Transcription Factor Stripe
Coordinated locomotion of Drosophila larvae depends on accurate patterning and stable attachment to the cuticle of both muscles and proprioceptors (chordotonal organs). Unlike muscle spindles in mammals, the fly chordotonal organs are not embedded in the body-wall muscles. Yet, the contractile system (muscles and tendons) and the chordotonal organs constitute two parts of a single functional unit that controls locomotion, and thus must be patterned in full coordination. It is not known how such coordination is achieved. Here we show that the positioning and differentiation of the migrating chordotonal organs are instructed by Stripe, the same transcription factor that promotes tendon cell specification and differentiation and is required for normal patterning of the contractile system. Our data demonstrate that although chordotonal organs are patterned in a Stripe-dependent mechanism similarly to muscles, this mechanism is independent of Stripe activity in tendon cells. Thus, the two parts of the locomotive system use similar but independent patterning mechanisms that converge to form a functional unit. Stripe plays at least a dual role in chordotonal development. It is required within the ligament cells for terminal differentiation and proper migration, without which no induction of ligament attachment cells takes place. Stripe's activity is then necessary within the recruited cells for their differentiation as attachment cells. Similarly to the biphasic differentiation program of tendons, terminal differentiation of chordotonal attachment cells is associated with sequential activation of the two Stripe isoforms-Stripe B and Stripe A.
Spatial Regulation of Cell Adhesion in the Drosophila Wing is Mediated by Delilah, a Potent Activator of βPS Integrin Expression
In spite of our conceptual view of how differential gene expression is used to define different cell identities, we still do not understand how different cell identities are translated into actual cell properties. The example discussed here is that of the fly wing, which is composed of two main cell types: vein and intervein cells. These two cell types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these two alternative differentiation programs are still very roughly drawn. Here we identify the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, βPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of βPS integrin expression. In the absence of normal Dei activity the level of βPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on βPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required.
